Proteases secreted into the intestinal lumen degrade dietary proteins. The central hypothesis is that proteases are also signaling molecules that directly regulate cells by specifically interacting with members of a new family of G-protein coupled receptors: the proteinase-activated receptors. The proposal focuses on proteinase-activated receptor-2 (PAR-2), a putative receptor for luminal pancreatic trypsin, tumor-associated trypsins and mast cell tryptase, that is highly expressed by intestinal epithelial cells and epithelial-derived tumor cells. Specific Aim 1 examines mechanisms of activation of PAR-2 by proteases. PAR-2 will be mutated to define cleavage sites in the extracellular tail. Antibodies to these sites will be tested for their ability to prevent receptor cleavage and activation so that they can be used as PAR- 2 antagonists. Cleavage exposes as tethered ligand that binds and activates cleaved PAR-2. To identify critical residues, peptide analogues of the tethered ligand will be tested for activity, specifically and metabolic stability, and then used for physiological studies. Thus, Aim 1 will generate PAR-2 antagonists and agonists for functional studies. Specific Aim 2 defines physiological roles of PAR-2 in the intestine and in tumor cells. To give insight into function, PAR-2 will be localized in tissues by immunohistochemistry, with light and electron microscopy, and in situ hybridization. Functional expression of PAR-2 in intestinal epithelial cells and epithelial-derived tumor cells will be confirmed by examining mechanisms of signal transduction. Specific PAR-2 agonist peptides and antagonist antibodies will be used to determine the role of PAR-2 in regulating (a) prostaglandin secretion in the intestinal epithelium, where prostanoids are of critical importance for cytoprotection, inflammation and local paracrine control mechanisms; and (b) growth of epithelial cells and epithelial-derived tumor cells. Signaling mechanisms regulating eicosanoid metabolism and growth will be examined. Specific Aim 3 investigates mechanisms that regulate these physiological responses to PAR-2. Comparisons of mechanisms of desensitization, resensitization and intracellular trafficking will be made between PAR-2, which is irreversibly activated by proteolysis, and the neurokinin 1 receptor, which is reversibly activated by binding substance P. Intracellular domains of PAR-2 and neurokinin 1 receptor will be exchanged to identify motifs that are important for lysosomal targeting of cleaved PAR-2 and recycling of internalized neurokinin 1 receptor. Together, these experiments will define new mechanisms of cellular regulation by proteases.